1. Field of the Invention
The present subject matter relates to a composition for reprogramming somatic cells to generate embryonic stem cell-like cells, comprising Bmi1 and a low molecular weight substance, and a method for generating embryonic stem cell-like cells using the same. In particular, the present subject matter relates to a composition for reprogramming somatic cells to generate embryonic stem cell-like cells by introducing Bmi1 into the somatic cells and treating the cells with low molecular weight substances including a MEK inhibitor and a GSK inhibitor, and a method for generating embryonic stem cell-like cells using the same.
2. Description of the Related Art
Unlike normal somatic cells, stem cells have self-renewal, that is, the ability to go through numerous cycles of cell division while maintaining a state of undifferentation, and potency, that is, the capacity to differentiate into specialized cell types under suitable conditions. Potency specifies the differentiation potential of the stem cells, and is typically divided into pluripotency, multipotency and unipotency. Therefore, the technique of allowing the stem cells to undergo self-renewal in cell cultures and transforming them into specialized cells has high potential in the cell therapy of various diseases.
Various stem cells including hematopoietic stem cells, bone marrow stem cells and neural stem cells are present in adults and can be isolated from the patients themselves and thus can be used in medical therapies without inducing immune rejection response. Cell therapy with adult stem cells solves the difficulty of securing donors for organ implantation.
However, adult stem cells have been so far known to remain multipotent. That is, tissue-specific stem cells are able to differentiate into a number of cells, but only those of a closely related family of cells. Disclosed in many reports are the effects that stem cells isolated from the central nervous system (Science 255, 1707-1710 1992; Science 287, 1433-1438 2000), the bone marrow (Science 276, 71-74, 1997; Science 287, 1442-1446, 2000; Science 284, 143-147, 1999), the retina (Science 287, 2032-2036, 2000) and the skeletal muscle (Proc. Natl. Acad. Sci. USA 96, 14482-14486, 1999; Nature 401, 390-394, 1999) undergo differentiation into the cells of closely related tissue. For example, hematopoietic stem cells can be differentiated into blood-related cells, neural stem cells into neurons or glial cells, and bone marrow stem cells into mesodermal cells. Moreover, adult stem cells, although able to theoretically undergo infinite self-renewal, have been reported with regard to difficulty in proliferating them in vitro. It is practically difficult to isolate a number of cells from patients.
Pluripotent stem cells are a wonderful resource overcoming the drawbacks of adult stem cells. Pluripotent stem cells can differentiate into nearly any cell and are allowed to replicate infinitely in vitro. Among the pluripotent stem cells known thus far are embryonic stem cells, embryonic germ cells and embryonic carcinoma cells, with most studies focusing on using embryonic stem cells for the purposes of differentiation into specific cells, functionality in animal models of diseases, and therapeutic potency for various diseases.
Nonetheless, the clinical use of embryonic stem cells, like adult stem cells, encounters barriers that must be overcome. Above all, because isolating embryonic stem cells results in the death of the fertilized human embryo, this raises ethical issues. Also, there is the problem of immunological rejection when differentiated cells derived from embryonic stem cells are implanted into patients.
A variety of approaches have been suggested to the above-mentioned problems, of which reprogramming differentiated cells into pre-differentiated cells has attracted the most attention. Reprogramming is a generic term expressing the induction of differentiated cells to dedifferentiate into pluripotent stem cells such as embryonic stem cells, generally achieved by 1) nuclear transfer, 2) cell fusion, 3) cell extract treatment, and 4) dedifferentiation technology for induced pluripotent stem cell (iPS cell) (Cell 132, 567-582, 2008).
iPS cell technology has succeeded in generating cells closer to embryonic stem cells than has any other technology. Since 2006 when iPS cells were first produced, a significant number of research articles have been issued. In principle, stem cells similar to embryonic stem cells, e.g., iPS cells, are established by transfection of four genes (reprogramming inducing genes; Oct4, Sox2, Klf4, and C-Myc/Oct4, Sox2, Nanog, Lin28) into mouse or human somatic cells, followed by culturing for a long period of time under conditions specialized for embryonic stem cells. These iPS cells have been demonstrated to resemble embryonic stem cells in their gene expression profile, epigenetics, in vitro/in vivo differentiation into all three germ layers, teratoma formation, chimeric mouse generation and the chimeric mouse's competency for germline transmission (Cell 126, 663-676, 2006; Science 318, 1917-1920, 2007).
However, too many gene factors used in reprogramming have made it difficult to understand the molecular mechanisms underlying reprogramming. To realize the full potential of iPS cells in practical clinical use, it will be essential to improve the reprogramming technology, although established, and to evaluate each generated iPS cell line for safety and efficacy.
Recent research reports have it that the inactivation of the tumor suppressor gene p53 markedly increases the efficiency of iPS (Nature 460, 1132-1135, 2009). p19Arf and p16Ink4a, both encoded by alternative reading frames of Arf/Ink4a locus, are known to induce the expression of p53 and Rb, respectively. By reducing the expression of both p16Ink4a and p19Arf, iPS cell formation was increased relative to that attained by reducing the expression of p19Arf alone (Nature, 460, 1140-1144, 2009).
Polycomb group (PcG) proteins are epigenetic gene silencers. Bmi1, one of the PcG proteins, is involved in the down-regulation of both p16Ink4a and p19Arf, which leads to suppressing the expression of p53 and Rb (Genes Dev, 2678-2690, 1999). Further, Bmi1 is known to regulate the expression of target genes by modifying chromatin organization. Thanks to these functions, Bmi1 plays an important role in the self-renewal of neural stem cells and hematopoietic stem cells. Based on this, the present inventors succeeded in the reprogramming of astrocytes to induce neural stem cells by overexpressing Bmi1 therein. The induced neural stem cells were similar in many aspects to those isolated from mice. Inter alia, the induced neural stem cells were found to have an increased expression level of Sox2, a gene essential for the self renewal of neural stem cells as one of reprogramming inducing genes (Biochem Biophys Res Commun. 371, 267-272, 2008).
Somatic cells require four (Oct4, Sox2, Klf4, C-Myc) or three (Oct4, Sox2, Klf4) genes for their dedifferentiation. It is known that these genes may not be additionally introduced into cells which endogenously express them. It was representatively demonstrated that the introduction of Oct4 alone induces the generation of iPS cells from mouse/human neural stem cells since they show the endogenous expression of Sox2, Klf4 and C-Myc (Nature, 461, 649-653, 2009).
It is reported that the addition of both the MEK inhibitor PD0325901 and the GSK3β inhibitor CHIR99021 can induce the differentiation of pre-iPS cells, which are in an intermediate state of the dedifferentiation process into fully reprogrammed cells (PLoS One, 6, 2237-2247, 2008).
In addition, the use of a G9a HMTase inhibitor and a DMNT inhibitor in a dedifferentiation process is known to increase the efficiency of reprogramming (Cell Stem Cell, 3, 568-574, 2008).
The efficiency of reprogramming can also be improved by treating with a histone deacetylase inhibitor (VPA) as part of a differentiation process (Cell Stem Cell, 4, 301-312, 2009).
Nowhere has, however, the induction of dedifferentiation by introducing a Bmi1 gene and treating with a low molecular weight substance and a method for generating pluripotent embryonic stem cell-like cells been known in the art.